Protooncogene Ras has become a central focus in many studies of neoplasia for a variety of reasons. Among these are the finding that Ras plays an important role in regulating cell proliferation (Berridge, M. J. et al. Biochem. J. 230: 345. 1984) and the frequency of the Ras mutations in many tumors; about 50% of human colon carcinomas and 90% of pancreatic carcinomas produce mutant Ras protein. (Varmus, H. E. Ann. Rev. Genet. 18: 553.1984. Farr, C. L. et al. Proc. Natl. Acad. Sci. 85: 1629. 1988. Barbacid, M. Ann. Rev. Biochem. 56: 779. 1997). This data has led to the widely-held belief that Ras proteins play a critical role in the control of carcinogenesis and inhibition of Ras may lead to the inhibition of cancer cell growth. Accordingly, much interest has been directed toward the study of Ras biochemistry, Ras mutation and any protein which might influence the biological activity of Ras.
Ras functions as a signal transducer for cell proliferation. Located on the inner surface of cell membrane, Ras is able to delay proliferative signal from growth factor receptors to the nucleus for initiation of cell growth. Mutations which cause hyperactivity of Ras may lead to cell transformation, which has been shown to occur in many human tumors. (Barbacid, M. Ann. Rev. Biochem. 56: 779.1987).
As with other guanine nucleotide-binding proteins, Ras is thought to be biologically active when bound to GTP. Its intrinsic GTPase converts Ras GTP to Ras GDP, which is thought to be biologically inactive. In our study, we have isolated a protein designated as p10 which, in the presence of cAMP, strongly inhibited H-Ras bound to GTP in vitro, an activity which inactivates the biological function of Ras. Whereas, in the absence of cAMP, p10 inhibited the intrinsic GTPase activity of Ras without affecting its GTP binding activity in vitro, which keeps Ras at its active form and activates Ras biologically. These results show that p10 activates or inhibits Ras in a cAMP dependent manner leading to the stimulation or inhibition of cell proliferation. It is well known that cyclic Adenosine monophosphate (cAMP) plays a critical role in determining if the cell will stay in the G0 phase or progress to the G1 phase [Boyntor, H. L. et al., Adv. Cyclic Nucleo. Res. 15: 193 (1983). Bannai, S. and Sheppard, J. R., Nature 250: 62 (1974). Bombik, B. M. and Burger, M., Exp. Cell Res. 80: 88 (1973). Boynton, A. L. and Whitfield, J. F., J. Cell Physiol. 101: 139 (1979). Boynton, A. L. et al., Life Sci. 22: 703 (1978). Burger, M. M. et al., Nature New Biol. 239: 161 (1972). Otten, J. et al., Biochem. Biophys. Res. Commun. 44: 1192 (1971). Otten, J. et al., J. Biol. Chem. 247: 7082 (1972). Rechler, M. M. et al., Exp. Cell Res. 104: 411 (1977). Rochette-Egly, C. et al., J. Cyclic Nucleotide Res. 5:385 (1979). Sheppard, J. R., Nature New Biol. 236: 14 (1972). L'Allemain, G. et al., Oncogene 14: 1981 (1997). Southgate, K. et al., Atherosclerosis 82: 113 (1990). Tominson, P. R. et al., Biochem Phamacol. 49: 1809 (1995). Indolfi, C. et al., Nat. Med. 3:775 (1997). Kram, R. et al., Proc. Natl. Acad.Sci. U.S.A. 270: 1432 (1973). Willingham, M. C. et al., Biochem. Biolphys. Res. Commun. 48: 743 (1972). Hogan, B. et al., Cell 2: 229 (1974). Pardee, A. B., Proc.Natl. Acad. Sci. U.S.A. 71: 1286 (1974). Lehnert, S., Exp. Cell Res. 121: 383 (1979). Rozengurt, E. and Pardee, A. B., J. Cell Physiol. 80: 273 (1972). Paul, D. and Walter, S., J. Cell Physiol. 85: 113 (1975). Froehlich, J. E. and Rachmeler, M., J. Cell. Biol. 55: 19 (1972). Froehlich, J. E. and Rachmeler, M., J. Cell. Biol. 60: 249 (1974). Lingwood, C. A. and Thomas, D. B., J. Natl. Cancer Res. 52: 1659 (1974). Frank, W., Exp. Cell Res. 71: 238 (1972). Cho. B. K. and Rose, N. R., Exp. Cell Res. 83: 261 (1974)]. The elevated cAMP level caused by serum starvation is necessary to keep the cell in the quiescent state (G0 state); whereas serum stimulation of the quiescent cell causes the decrease of intracellular cAMP levels which is a signal for the G0 to G1 transition leading to the resumption of cell cycle. On the other hand, protoncogene Ras was found to be a key molecule controlling the cell to leave GO state (Peeper, D. S. et al., Nature 386: 177 (1997). Dobrowolski, S. et al., Mol. Cell Biol. 14: 5441 (1994)). In the present studies, we found that in the presence of cAMP, p10 functionally inhibits Ras, whereas, in the absence of cAMP, p10 functionally activates Ras. In response to serum starvation, the elevated cAMP levels render p10 to inhibit Ras (by inhibiting Ras GTP binding) causing the cell to enter a quiescent or G0 state. Serum stimulation of a quiescent cell, on the other hand, causes the decrease of intracellular cAMP levels which render p10 to activate Ras (by inhibiting Ras GTPase activity) leading to the G0 to G1 transition and resumption of the cell cycle.
This invention describes the methods for isolation of p10 protein which activates/inhibits Ras in a cAMP dependent manner as well as the biochemical activity of p10 found in our laboratory at Ambryx.